Production and Characterization of Recombinant Single-Chain Variable Fragment (scFv) Antibody Against Fasciola...

Saposin-like protein 2 (SAP2) exhibits strong immunogenicity as an antigen for immunodiagnosis in ruminant and human fasciolosis. Most available immunodiagnostic test kits are based on polyclonal and monoclonal antibodies against antigens from Fasciola spp. Previous studies demonstrated that polyclonal and monoclonal antibodies against SAP2 showed high specificity and could effectively detect Fasciola spp. infections at an early stage. However, polyclonal antibodies are extremely difficult to produce, and quality control is not possible during production; the procedure also involves considerable financial investment. To overcome these problems, we developed a single-chain variable fragment (scFv) to control quality in each production cycle and reduce the cost of manufacturing immunodiagnostic kits. Our objectives were to produce and characterize an scFv that binds the SAP2 from the liver fluke Fasciola gigantica. We constructed the scFv by genetic engineering: we cloned immunoglobulin genes and linked them with flexible polypeptide linkers composed of repeating glycine and serine residues. We selected an scFv with high affinity for binding SAP2 using the phage-display technique and produced it using a prokaryotic expression system. The scFv was characterized via in silico and in vitro methods to confirm its specificity for SAP2, including IMGT/V-QUEST, IMGT/Collier-de-Perles, HADDOCK 2.4, ELISA, immunoblotting, and immunohistochemistry. The scFv was successfully produced and purified using Ni-NTA affinity chromatography. The purified scFvFgSAP2 was approximately 27 kDa, as confirmed by SDS-PAGE and immunoblot analysis. An indirect ELISA and immunoblotting indicated that scFvFgSAP2 had strong reactivity with F. gigantica compared to other parasite species. Moreover, immunolocalization of scFvFgSAP2 confirmed that it binds specifically to natural SAP2 in the cecal epithelium cells of F. gigantica. Therefore, this scFv targeting SAP2 is an effective material and can be used to develop immunodiagnostic procedures.